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Picosecond Laser Processing of Photosensitive Glass for Generation of Biologically Relevant Microenvironments applied sciences Article Picosecond Laser Processing of Photosensitive Glass for Generation of Biologically Relevant Microenvironments Florin Jipa 1, Stefana Orobeti 1, Cristian Butnaru 1, Marian Zamfirescu 1, Emanuel Axente 1 , Felix Sima 1,2,* and Koji Sugioka 2,* 1 CETAL, National Institute for Laser, Plasma and Radiation Physics (INFLPR), 409 Atomistilor, RO-77125 Magurele, Romania; florin.jipa@inflpr.ro (F.J.); stefana.iosub@inflpr.ro (S.O.); cristian.butnaru@inflpr.ro (C.B.); marian.zamfirescu@inflpr.ro (M.Z.); emanuel.axente@inflpr.ro (E.A.) 2 RIKEN Center for Advanced Photonics, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan * Correspondence: felix.sima@inflpr.ro (F.S.); [email protected] (K.S.); Tel.: +40-21-457-4550 (F.S.) Received: 28 October 2020; Accepted: 12 December 2020; Published: 15 December 2020 Abstract: Various material processing techniques have been proposed for fabrication of smart surfaces that can modulate cellular behavior and address specific clinical issues. Among them, laser-based technologies have attracted growing interest due to processing versatility. Latest development of ultrashort pulse lasers with pulse widths from several tens of femtoseconds (fs) to several picoseconds (ps) allows clean microfabrication of a variety of materials at micro- and nanoscale both at surface and in volume. In this study, we addressed the possibility of 3D microfabrication of photosensitive glass (PG) by high repetition rate ps laser-assisted etching (PLAE) to improve the fabrication efficiency for the development of useful tools to be used for specific biological applications. Microfluidic structures fabricated by PLAE should provide the flow aspects, 3D characteristics, and possibility of producing functional structures to achieve the biologically relevant microenvironments. Specifically, the microfluidic structures could induce cellular chemotaxis over extended periods in diffusion-based gradient media. More importantly, the 3D characteristics could reproduce capillaries for in vitro testing of relevant organ models. Single cell trapping and analysis by using the fabricated microfluidic structures are also essential for understanding individual cell behavior within the same population. To this end, this paper demonstrates: (1) generation of 3D structures in glass volume or on surface for fabrication of microfluidic channels, (2) subtractive 3D surface patterning to create patterned molds in a controlled manor for casting polydimethylsiloxane (PDMS) structures and developing single cell microchambers, and (3) designing glass photo-masks to be used for sequel additive patterning of biocompatible nanomaterials with controlled shapes, sizes, and periodicity. Mesenchymal stem cells grown on laser-processed glass surfaces revealed no sign of cytotoxicity, while a collagen thin coating improved cellular adhesion. Keywords: picosecond laser-assisted etching; photosensitive glass; 3D microfluidics; PDMS; microchambers; photo-masks; biomimetics 1. Introduction Biochips, such as microfluidic, lab-on-a-chip, and organ-on-a-chip devices, and micro total analysis systems have opened a new door for medical and biological investigations due to capability of generating biologically relevant microenvironments and potential of single cell analysis [1,2]. Generally, shape-controlled micropatterns grown on solid substrates by additive manufacturing are adequate for the generation of biomimetic cell-instructive environments, able to modulate specific cell responses for applications in tissue engineering, regenerative medicine, or cancer therapy. These surfaces and cell interfaces are needed for the next generation of biomaterials [3]. For example, in the cancer therapy, Appl. Sci. 2020, 10, 8947; doi:10.3390/app10248947 www.mdpi.com/journal/applsci Appl. Sci. 2020, 10, 8947 2 of 17 studies have shown that tumor cells may exhibit ‘plasticity’ in response to microenvironmental cues [4]. Interfacial geometry that dictates cancer cell tumorigenicity has revealed how the characteristics of micropatterns (e.g., size, shape, and perimeter characteristics) influence a population of tumor cells that can further guide cancer cells toward a stem cell-like state [5]. A clinical biochip consisting of more than 1000-nanoliter-sized microchambers was developed for single immune cell investigation [6]. The proteins secreted by individual cells were then captured by antibody stripes followed by multiplexed fluorescent detection. Such bioplatform proved successful ex vivo quantification of the polyfunctional diversity of tumor antigen-specific T lymphocytes. New biochip platforms consisting of microwell arrays were recently proposed for cell-based immunological studies with various applications related to immune cell secretion, cytotoxicity, migration, phenotyping, and immune cell engineering [7]. In addition, high-throughput cell trapping assays with single cell accuracy were efficiently developed into microfluidic lab-on-chip systems [8]. Meanwhile, lasers are increasingly getting important as tools for biomaterial modification or fabrication of biochips. The main advantages of laser techniques in comparison to other approaches are extensively addressed in the literature [9,10]. One can mention the high processing versatility of a variety of materials together with fast, clean, flexible, and controllable microfabrication capability. During the last decade, laser technologies were explored in the field of biomimetics with new cutting-edge applications [11]. The state of the art of “Laser Technology in Biomimetics” covering a broad spectrum of materials, methodologies, and applications was comprehensively reported in [12]. This book covers aspects of laser–matter interaction of nanosecond pulsed lasers for 2D patterning using well-known deposition techniques, such as pulsed laser deposition (PLD) and matrix-assisted pulsed laser evaporation (MAPLE) for material coating and ultrashort laser pulses for 3D modification of materials. PLD and MAPLE employ pulsed laser beams, typically nanosecond laser pulses, to ablate solid targets in a vacuum. Then, the ablated material is deposited onto substrates opposed to the targets to form thin coatings. Different from PLD, which is used for inorganic coatings only, the MAPLE process employs milder conditions (laser energies one order of magnitude lower) adequate for coating any inorganic, organic, and hybrid materials with high versatility. Thus, the safe transfer and deposition of delicate compounds such as proteins and biopolymers are possible without affecting stability and deteriorating functionalities [11]. In addition, graphene oxide (GO) and GO nanocolloids (GON) can be deposited by MAPLE, which are very challenging matrices for drug release to target cancer cells. It has been very recently found that the MAPLE process provides safe concentration windows of GON for five different human melanoma cell lines as well as for nontransformed melanocytes and human dermal fibroblasts [13]. A specific feature of laser–matter interaction, in particular when using ultrashort pulse lasers with pulse widths of several tens of femtosecond (fs) to several picoseconds (ps), offers the possibility to precisely control three-dimensional (3D) micro-/nanofabrication in transparent materials [14–20]. Indeed, tightly focused ultrashort pulse lasers can achieve very high peak intensities ( 10 TW cm 2), ≈ − resulting in strong nonlinear absorption phenomena in transparent materials to allow localized modification in volume with high precision [21]. The fundamental mechanisms of fs laser pulse interaction with the transparent materials were extensively explored in the literature [15,17,19,21]. Some of the involved processes are competitive at different time scales and are strongly dependent not only on the laser irradiation parameters (pulse energy, pulse duration, laser wavelength, repetition rate, and focusing optics) but also on the physical-chemical properties of materials processed. The extensive studies of the laser–matter interaction together with new development of high-precision 3D CAD-CAM (computer-aided design/computer-aided manufacturing) stages potentiate the unique characteristics of ultrashort pulse lasers for 3D micro- and nanofabrication of the diverse materials. Advanced setups consisting of ultrashort pulse lasers and high-precision 3D translation stages have been developed for selective and precise generation of complex 3D microstructures embedded inside photosensitive glasses (PGs). The process is known as fs laser-assisted etching (FLAE) and Appl. Sci. 2020, 10, 8947 3 of 17 was continuously optimized for concrete applications [22–27]. It begins with laser direct writing (LDW) of PG in which the stage scanning speed, focusing position, and laser power are adjusted to achieve the desired 3D shape structure. An initial annealing treatment follows the laser irradiation, by which Ag atoms generated by the laser irradiation are clustering to form nuclei for the growth of a crystalline phase of lithium metasilicate at the laser-exposed regions. The wet chemical etching using hydrofluoric (HF) acid solution is then applied for selective removal of the crystalline phase to create 3D hollow structures. Consequently, microfluidic platforms with scale-down (µm) and scale-up (mm) configurations can be achieved. A typical PG used with FLAE is commercially available Foturan glass. It exhibits high chemical stability, high transparency for optical interrogation, and biocompatibility. Advanced laser processing protocols of Foturan glass were described in details
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